Investigational Studies Of Small-molecule Compounds Specifically Targeting The Akt-Qct4 Regulatory Pathway Of Cancer Stem Cells

The incidence and mortality of cancer has been increasing all over the world, making it the leading cause of death. Ample evidence has established that tumorigenesis (or carcinogenesis) results from complex interactions involving multiple factors, multiple genes at multiple stages, and its incurability is mainly caused by metastasis, drug resistance, and radiation resistance.Many studies have focused on the similarities between tumorigenesis and embryogenesis. With the rapid progress of stem cell researches, the hypothesis of cancer stem cells is gaining increasing attention. According to this hypothesis, tumor cells are heterogeneous and only a subpopulation of them termed "cancer stem cells" has the ability to proliferate and self-renewal extensively, to give rise to differentiated cells, and to form new tumors, and cancer stem cells play an important role in the processes of tumor development and metastasis.Oct4 is a transcription factor encoded by POU5F1 gene. It is mainly expressed in embryonic stem cells, germline stem cells, and embryonal carcinoma cells, and plays an indispensible role in maintaining the pluripotency and self-renewal of these pluripotent stem cells. Accumulating literature has documented the detection of Oct4 mRNA and protein in a variety of cancer cell lines and solid tumor specimens, but Oct4’s mechanism of action in differentiated cells or tissues remains largely unclear.Our previous work has established that Oct4 can be phosphorylated by Akt which can promote the self-renewal and tumorigenicity of embryonic carcinoma cells. Furthermore, unphosphorylated Oct4 preferentially bound to the AKT1 promoter to repress Akt transcription, and Akt-mediated phosphorylation of Oct4 at T235 (Oct4-pT235) caused its disassociation from the AKT promoter, thereby activating Akt transcription in a positive feedback manner to form the "Akt-Oct4 regulatory circuit" and promote cell survival.In this thesis, I asked the question about "if the Akt-Oct4 regulatory circuit is over-activated in cancer stem cells and if it can serve as a therapy target for drug intervention". The expression of Oct4 and Oct4-pT235 in human somatic cancer cell lines and tissues was examined using combinatory approaches. Oct4-pT235 was found predominantly localized in the nucleus of cancer cells. The level of Oct4-pT235 was increased in glioblastoma stem-like cells. The PI3K-Akt signaling pathway and the major stem cell self-renewal pathways were upregulated in glioblastoma stem-like cells, and this was associated with an increased Oct4-pT235 level in those cells. Notably, inhibiting Akt activities by Akti-1/2 decreased the Oct4-pT235 level and attenuated the proliferation of glioblastoma stem-like cells.A previous study has shown that tranilast, the synthetic agonist of the aryl hydrocarbon receptor (AhR), can downregulate the expression of Oct4 in stem-like breast cancer cell lines and inhibit their proliferation and metastasis by an unidentified mechanism. In this thesis, I focused on the effects of ITE, a tryptophan derivative and natural ligand of AhR, on Oct4 expression. Unlike the normoxia condition where endogenous ITE was detectable when U87 cells were grown in tryptophan-rich DMEM, the ITE level was reduced under hypoxic conditions. In parallel, the Oct4 transcription level in hypoxia-treated cells was elevated compared with normoxia-treated cells. Consequently, ITE suppressed Oct4 transcription and decreased the level of Oct4-pT235 and Oct4. Synthetic ITE induced the differentiation of stem-like cancer cells and reduced the tumorigenic potential in subcutaneous xenograft tumor models.Metformin, a marketed drug in treating Type II diabetes, has recently been shown to reduce the expression of Oct4 at the transcription level. Towards a possible dual inhibition of Oct4 at both transcriptional and post-translational levels, I attempted to examine the effects of combining Akti-1/2 and metformin against glioblastoma stem-like cells. The combination of Akti-1/2 and metformin significantly reduced the level of Oct4-pT235 and Oct4 while concomitantly decreasing the Akt-pT308 and Akt-pS473 levels. Moreover, there was a reciprocal suppressive interaction between AMPK and Akt. Akti-1/2 reduced AKT to de-repress the AMPK inhibition and metformin activated AMPK. The results indicated that the two compounds dually targeted the Akt-Oct4 regulatory circuit and synergistically attenuated the proliferation of glioblastoma stem-like cells in in vitro culture system and subcutaneous xenograft tumour models.Taken together, my thesis work reported here explored the possibility of targeting the Akt-Oct4 regulatory circuit in stem-like cancer cells. Inhibition of Akt activities effectively decreased the amount of Oct4-pT235 at the post-translational level and attenuated the proliferation of glioblastoma stem-like cells. ITE inhibited Oct4 transcription, induced the differentiation of stem-like cancer cells and attenuated their proliferation, and thus may serve as a promising lead compound for combating human cancers. Akt inhibitor Akti-1/2 combined with metformin exerted synergistic and remarkable anti-proliferative effects against glioblastoma stem-like cells, most likely by targeting the Akt-Oct4 regulatory circuit, and therefore the combination therapy of Akti-1/2 plus metformin may represent a novel and promising strategy for treating human glioblastoma.